Viral vector production system

Abstract

The present invention relates to a nucleic acid sequence comprising a binding site operably linked to a nucleotide of interest, wherein the binding site is capable of interacting with an RNA-binding protein such that translation of the nucleotide of interest is repressed in a viral vector production cell.

Description

[0001]This application is the national stage under 35 U.S.C. 371 of International Application No. PCT / GB2014 / 053813, filed on Dec. 19, 2014, and claiming priority to GB Application No. 1322798.8. filed on Dec. 20, 2013, both of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention relates to the production of viral vectors. More specifically, the present invention relates to modification of the translation of a nucleotide of interest which is encoded by a viral vector, in a viral vector production cell.BACKGROUND TO THE INVENTION[0003]Gene therapy broadly involves the use of genetic material to treat disease. It includes the supplementation in cells with defective genes (e.g. those harbouring mutations) with functional copies of those genes, the inactivation of improperly functioning genes and the introduction of new therapeutic genes.[0004]Therapeutic genetic material may be incorporated into the target cells of a host using vectors to enable the tran...

AI Technical Summary

Benefits of technology

[0022]We have shown that this problem can be overcome by using a heterologous translation control system in eukaryotic cell cultures to repress the translation of the NOI and thus repress or prevent expression of the protein encoded by the NOI. We have surprisingly found that use of this system does not impede the production of packageable vector genome molecules nor the activity of vector virions, and does not interfere with the long-term expression of the NOI in the target cell.

[0023]In one aspect, the present invention provides a nucleic acid sequence comprising a binding site operably linked to a nucleotide of interest, wherein the binding site is capable of interacting with an RNA-binding protein such that translation of the nucleotide of interest is repressed or prevented in a viral vector production cell.

[0042]We demonstrate for the first time the use of a bacterial translation repressor system to increase RNA (retroviral) vector titres in eukaryotic cell production systems by repressing translation of the NOI mRNA. The placement of a binding site for an RNA binding protein (e.g. a TRAP-binding sequence, tbs) upstream of the NOI translation initiation codon allows specific repression of translation of mRNA derived from the internal expression cassette, whilst having no detrimental effect on production or stability of vector RNA. We have also shown that production and function of retroviral vectors appeared to be minimally affected by the presence of the tbs in the vector genome. This implies that either reverse transcriptase (RT) can displace TRAP from the tbs or that TRAP is not efficiently bound to the tbs in the context of the long vector genome RNA packaged within the virion. We have also shown that a similar configuration of tbs placed within the NOI expression cassette of DNA viral vectors, such as AAV, allows NOI translation repression by TRAP whilst maintaining production of active viral vector virions.

[0046]The RNA-binding protein (e.g. TRAP) open-reading frame may be codon-optimised for expression in mammalian (e.g. Homo sapiens) cells, since the bacterial gene sequence is likely to be non-optimal for expression in mammalian cells. The sequence may also be optimised by removing potential unstable sequences and splicing sites. The use of a HIS-tag C-terminally expressed on the TRAP protein appears to offer a benefit in terms of translation repression and may optionally be used. This C-terminal HIS-tag may improve solubility or stability of the TRAP within eukaryotic cells, although an improved functional benefit cannot be excluded. Nevertheless, both HIS-tagged and untagged TRAP allowed robust repression of transgene expression. Certain cis-acting sequences within the RNA-binding protein (e.g. TRAP) transcription unit may also be optimised; for example, EF1a promoter-driven constructs enable better repression with low inputs of TRAP plasmid compared to CMV promoter-driven constructs in the context of transient transfection.

Problems solved by technology

A significant limitation of producing therapeutic retroviral vectors at high or even moderate titres can be the adverse expression of the protein encoded by the transgene / NOI in production cells, particularly when the transgene is driven by a powerful, constitutive promoter.

Tissue-specific promoters to silence transgene expression in production cells have been used as a solution to this problem but a significant drawback with this approach is that often gene expression from tissue specific promoters is not as robust as it is from constitutive ones, and can be less predictable or more variable in the different animal models that are utilised during vector development.

This implies that either reverse transcriptase (RT) can displace TRAP from the tbs or that TRAP is not efficiently bound to the tbs in the context of the long vector genome RNA packaged within the virion.

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